Human p53-proficient osteosarcoma U2OS and p53 deficient lung non-small cell carcinoma H1299 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere as described previously (He et al, 2016 (link); Sun et al, 2012 (link)). Flag-SENP1, its catalytically-inactive C603S mutant, V5-SENP1, GST-SENP1 (WT and the C603S mutant), His-SUMO1, His-SUMO2 plasmids as well as the p53 expressing plasmids were previously described (Sun et al., 2012 (link); Sun et al, 2018 (link)). Anti-p53 (Do-1, Santa Cruz Biotechnology), anti-MDM2 (SMP14, Santa Cruz Biotechnology), anti-p21 (Ab-11, NeoMarkers), anti-Flag (M2, Sigma), anti-V5 (Invitrogen), and anti-SENP1 (abcam) were purchased.
Protocol detail
Studying p53 Regulation in Cancer Cells
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Atmosphere
Cells
Eagle
Fetal bovine serum
Human
Lung non small cell carcinoma
Mdm2
Osteosarcoma
Penicillin
Plasmids
Senp1
Streptomycin
Sumo1
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Variable analysis
independent variables
- Flag-SENP1
- Flag-SENP1 C603S mutant
- V5-SENP1
- GST-SENP1 (WT and C603S mutant)
- His-SUMO1
- His-SUMO2
- P53 expressing plasmids
- Expression levels or activity of p53, MDM2, and p21
- Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, and 0.1 mg/ml streptomycin
- Cell lines: Human p53-proficient osteosarcoma U2OS and p53 deficient lung non-small cell carcinoma H1299
- Incubation conditions: 37 °C in a 5% CO2 humidified atmosphere
- Negative control: Catalytically-inactive C603S mutant of SENP1
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